Core-shell capsules for encapsulation of particles, colloids, and cells

ABSTRACT

Methods for preparing capsules, such as micro- and/or nanocapsules from all-aqueous emulsions are described herein. The method includes mixing, combining, or contacting a first electrically charged phase containing a first solute with at least an optionally charged second phase containing a second solute. The solutes are incompatible with each other. The electrostatic forces between the two solutions induce the formation of droplets of a dispersed phase in a continuous phase. The droplets are then solidified to form the capsules.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/564,372, filed Dec. 9, 2014, which claims priority to and benefit ofU.S. Provisional Application No. 61/913,660, filed Dec. 9, 2013.Application Ser. No. 14/564,372, filed Dec. 9, 2014, and Application No.61/913,660, filed Dec. 9, 2013, are hereby incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

This invention is in the field of micro- and nanocapsules prepared fromall-aqueous emulsions.

BACKGROUND OF THE INVENTION

Hydrogel capsules have been used to encapsulate cells since they canallow facile diffusion of oxygen and nutrients to the cells. Suchcompositions have been developed as potential therapeutics for a rangeof diseases including type I diabetes, cancer, and neurodegenerativedisorders such as Parkinson's.

Traditional processing of hydrogel particles, such as alginateparticles, provides little control over the microstructure or size(diameter) of the capsules. When living cells are encapsulated in theparticles, the diffusion of oxygen and nutrients is restricted by thepresence of the thick hydrogel shell. Also, proliferation and fusion ofcells are prohibited due to the lack of aqueous space.

Moreover, the preparation of such particles often involves the use oforganic solvents. Such solvents are costly, toxic, flammable and harmfulto the environment. Upon solidifying the dispersed phase to form thesolid capsules, organic solvents must be extracted by repeated washingwhich is time consuming. Therefore, it is highly desirable to replacethe organic solvents with all aqueous solute to avoid these tedioussteps to remove the organic phases. In addition, when protein solutionsare exposed to the oil phase, denaturation of proteins often occurs atthe water-oil (w/o) interface, reducing the bioactivity of the proteins.

Recent approaches to produce core-shell hydrogel capsules without theneed for organic solvents using miscible aqueous solutions typicallyresulted in leakage of the payload.

Therefore, there exists a need for improved methods for preparinghydrogel capsules, particularly capsules that prevent leakage of thepayload to be encapsulated but allows for efficient passage of oxygenand other nutrients to facilitate cell growth/survival.

SUMMARY OF THE INVENTION

Methods for preparing capsules, such as micro- and/or nanocapsules, fromall-aqueous emulsions are described herein. The method includes mixing,combining, or contacting a first electrically charged phase containing afirst solute with at least an optionally charged second phase containinga second solute. The solutes are incompatible with each other. Theelectrostatic forces between the two solutions induce the formation ofdroplets of a dispersed phase in a continuous phase. The droplets arethen solidified to form the capsules.

In some embodiments, a core-shell structured emulsion can also begenerated with the all-aqueous electrospray approach. A round capillarywith a tapered nozzle can be coaxially inserted into another taperedsquared capillary, forming a co-flowing geometry. Two immiscible aqueousphases are separately injected into the inner and outer glasscapillaries, forming an inner phase-in-outer phase jet. The outer(shell) phase can be charged by a high-voltage power supply and thecompound jet is forced to go through a ring-shaped counter electrodeunder electrostatic forces. Upon breakup of the jet, core-shellstructured droplets finally fall into the continuous phase. The relativesizes of the core and shell of the emulsion can be easily adjusted bychanging the flow rates ratios of the two fluids.

The capsules described herein can be used for a variety of applications,such as drug delivery (e.g., small molecules, biomolecules, cells, etc.)and encapsulation of active ingredients, such as proteins, insecticides,herbicides, salts, and macromolecules etc.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows formation of aqueous droplets. Panel a is a schematic ofthe formation of aqueous droplets in the all-aqueous electrosprayapproach and the passage of the droplets through a counter electrode.Panel b and panel c are optical images of the coaxial jet/droplets.

FIG. 2 is a graph showing the diameter of PEG-rich droplets (microns) asa function of the applied DC voltage (kV).

FIGS. 3A and 3B show formation of core-shell capsules. FIG. 3A is aschematic of the formation of aqueous droplets and the phase separationof the dextran shell and PEG core. FIG. 3B is a micrograph ofPEG/dextran core-shell capsules.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

“All-aqueous emulsion” and “Aqueous two-phase systems (ATPSs)” are usedinterchangeable and refer to an emulsion containing an aqueous dispersedphase in an aqueous continuous phase.

“Incompatible”, as used herein, generally means the binding energybetween two solute molecules of the same kind is lower than the bindingenergy between two different kinds of solute molecules.

The term “diameter” is art-recognized and is used herein to refer toeither of the physical diameter or the hydrodynamic diameter. Thediameter of emulsion typically refers to the hydrodynamic diameter. Thediameter of the capsules, both in spherical or non-spherical shape, mayrefer to the physical diameter in the hydrated state. The diameter ofthe particles, colloids and cells which are encapsulated inside thecapsules refers to the physical diameter in the hydrated state. As usedherein, the diameter of a non-spherical particle or a non-sphericalcapsule may refer to the largest linear distance between two points onthe surface of the particle. When referring to multiple particles orcapsules, the diameter of the particles or the capsules typically refersto the average diameter of the particles or the capsules. Diameter ofparticles or colloids can be measured using a variety of techniques,including but not limited to the optical or electron microscopy, as wellas dynamic light scattering.

The term “biocompatible” as used herein refers to one or more materialsthat are neither themselves toxic to the host (e.g., an animal orhuman), nor degrade (if the material degrades) at a rate that producesmonomeric or oligomeric subunits or other byproducts at toxicconcentrations in the host.

The term “biodegradable” as used herein means that the materialsdegrades or breaks down into its component subunits, or digestion, e.g.,by a biochemical process, of the material into smaller (e.g.,non-polymeric) subunits.

The term “microspheres” or “microcapsules” is art-recognized, andincludes substantially spherical solid or semi-solid structures, e.g.,formed from biocompatible polymers such as subject compositions, havinga size ranging from about one or greater up to about 1000 microns. Theterm “microparticles” is also art-recognized, and includes microspheresand microcapsules, as well as structures that may not be readily placedinto either of the above two categories, all with dimensions on averageof less than about 1000 microns. A microparticle may be spherical ornonspherical and may have any regular or irregular shape. If thestructures are less than about one micron in diameter, then thecorresponding art-recognized terms “nanosphere,” “nanocapsule,” and“nanoparticle” may be utilized. In certain embodiments, the nanospheres,nanocapsules and nanoparticles have an average diameter of about 500 nm,200 nm, 100 nm, 50 nm, 10 nm, or 1 nm.

A composition containing microparticles or nanoparticles may includeparticles of a range of particle sizes. In certain embodiments, theparticle size distribution may be uniform, e.g., within less than abouta 20% standard deviation of the mean volume diameter, and in otherembodiments, still more uniform, e.g., within about 10% of the medianvolume diameter. The term “capsule” as used herein refers tosubstantially spherical solid or semi-solid structures, e.g., formedfrom biocompatible polymers such as subject compositions.

The term “particle” as used herein refers to any particle formed of,having attached thereon or thereto, or incorporating a therapeutic,diagnostic or prophylactic agent, optionally including one or morepolymers, hydrogel-forming materials, liposomes micelles, or otherstructural material. A particle may be spherical or nonspherical. Aparticle may be used, for example, for diagnosing a disease orcondition, treating a disease or condition, or preventing a disease orcondition. A capsule is a form of particle. Unless the context indicatesotherwise, references herein to a particle are understood to includereference to a capsule.

The phrases “parenteral administration” and “administered parenterally”are art-recognized terms, and include modes of administration other thanenteral and topical administration, such as injections, and includewithout limitation intravenous, intramuscular, intrapleural,intravascular, intrapericardial, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradennal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrastemal injection and infusion.

The term “surfactant” as used herein refers to an agent that lowers thesurface tension of a liquid.

The term “therapeutic agent” refers to an agent that can be administeredto prevent or treat a disease or disorder. Examples include, but are notlimited to, a nucleic acid, a nucleic acid analog, a small molecule, apeptidomimetic, a protein, peptide, carbohydrate or sugar, lipid, orsurfactant, or a combination thereof.

The term “treating” preventing a disease, disorder or condition fromoccurring in an animal which may be predisposed to the disease, disorderand/or condition but has not yet been diagnosed as having it; inhibitingthe disease, disorder or condition, e.g., impeding its progress; andrelieving the disease, disorder, or condition, e.g., causing regressionof the disease, disorder and/or condition. Treating the disease orcondition includes ameliorating at least one symptom of the particulardisease or condition, even if the underlying pathophysiology is notaffected, such as treating the pain of a subject by administration of ananalgesic agent even though such agent does not treat the cause of thepain.

The phrase “pharmaceutically acceptable” refers to compositions,polymers and other materials and/or dosage forms which are, within thescope of sound medical judgment, suitable for use in contact with thetissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” refers topharmaceutically acceptable materials, compositions or vehicles, such asa liquid or solid filler, diluent, solvent or encapsulating materialinvolved in carrying or transporting any subject composition, from oneorgan, or portion of the body, to another organ, or portion of the body.Each carrier must be “acceptable” in the sense of being compatible withthe other ingredients of a subject composition and not injurious to thepatient.

The term “pharmaceutically acceptable salts” is art-recognized, andincludes relatively non-toxic, inorganic and organic acid addition saltsof compounds. Examples of pharmaceutically acceptable salts includethose derived from mineral acids, such as hydrochloric acid and sulfuricacid, and those derived from organic acids, such as ethanesulfonic acid,benzenesulfonic acid, and p-toluenesulfonic acid. Examples of suitableinorganic bases for the formation of salts include the hydroxides,carbonates, and bicarbonates of ammonia, sodium, lithium, potassium,calcium, magnesium, aluminum, and zinc. Salts may also be formed withsuitable organic bases, including those that are non-toxic and strongenough to form such salts. For purposes of illustration, the class ofsuch organic bases may include mono-, di-, and trialkylamines, such asmethylamine, dimethylamine, and triethylamine; mono-, di- ortrihydroxyalkylamines such as mono-, di-, and triethanolamine; aminoacids, such as arginine and lysine; guanidine; N-methylglucosamine;N-methylglucamine; L-glutamine; N-methylpiperazine; morpholine;ethylenediamine; and N-benzylphenethylamine.

The term “therapeutically effective amount” refers to an amount of thetherapeutic agent that, when incorporated into and/or onto particlesdescribed herein, produces some desired effect at a reasonablebenefit/risk ratio applicable to any medical treatment. The effectiveamount may vary depending on such factors as the disease or conditionbeing treated, the particular targeted constructs being administered,the size of the subject, or the severity of the disease or condition.One of ordinary skill in the art may empirically determine the effectiveamount of a particular compound without necessitating undueexperimentation. In some embodiments, the term “effective amount” refersto an amount of a therapeutic agent or prophylactic agent to reduce ordiminish the symptoms of one or more diseases or disorders of the brain,such as reducing tumor size (e.g., tumor volume) or reducing ordiminishing one or more symptoms of a neurological disorder, such asmemory or learning deficit, tremors or shakes, etc. In still otherembodiments, an “effective amount” refers to the amount of a therapeuticagent necessary to repair damaged neurons and/or induce regeneration ofneurons.

The terms “incorporated” and “encapsulated” refers to incorporating,formulating, or otherwise including an active agent into and/or onto acomposition that allows for release, such as sustained release, of suchagent in the desired application. The terms contemplate any manner bywhich a therapeutic agent or other material is incorporated into apolymer matrix, including for example: attached to a monomer of suchpolymer (by covalent, ionic, or other binding interaction), physicaladmixture, enveloping the agent in a coating layer of polymer, andhaving such monomer be part of the polymerization to give a polymericformulation, distributed throughout the polymeric matrix, appended tothe surface of the polymeric matrix (by covalent or other bindinginteractions), encapsulated inside the polymeric matrix, etc. The term“co-incorporation” or “co-encapsulation” refers to—the incorporation ofa therapeutic agent or other material and at least one other therapeuticagent or other material in a subject composition.

More specifically, the physical form in which any therapeutic agent orother material is encapsulated in polymers may vary with the particularembodiment. For example, a therapeutic agent or other material may befirst encapsulated in a microsphere and then combined with the polymerin such a way that at least a portion of the microsphere structure ismaintained. Alternatively, a therapeutic agent or other material may besufficiently immiscible in the polymer that it is dispersed as smalldroplets, rather than being dissolved, in the polymer.

II. Electrostatic Methods for Producing all Aqueous Emulsions

Methods for preparing all-aqueous emulsions, including stable emulsionsand/or emulsions having high viscosity and/or ultra-low interfacialtension are described herein. The method includes mixing, combining, orcontacting a first charged solution containing a first solute (e.g.,dispersed phase) with an optionally charged second solution containing asecond solute (e.g., continuous phase). The solutes are incompatiblewith each other. It has been discovered that the electrostatic forcesbetween the two solutions induce the formation of droplets of adispersed phase in a continuous phase.

The emulsions can be used to form micro- and nanocapsules, such ashydrogel capsules. In some embodiments, a coaxial jet is formed bycoflowing a first, core phase in a second, shell phase which contains ahydrogel-forming material. The jet is forced through a counter electrodeto break up the jet and form core-shell structured droplets. Thedroplets are solidified to form the capsules by inducing formation ofthe hydrogel.

A. Incompatible Solutes

Aqueous two-phase systems (ATPSs) are formed by dissolving twoincompatible solutes in water above the critical concentrations forphase separation. These incompatible solutes can redistribute in waterand form immiscible aqueous phases, if the reduction in enthalpy issufficient to overcome the energy cost associated with the increasedentropy. This often requires each solute species of an ATPS to interactmore strongly with itself than with the other species, leading to thesegregation of solute of the same species and phase separation.

A variety of solutes known in the art can be used to form theall-aqueous emulsions. Exemplary solutes include, but are not limitedto, polymers, such as polyethylene glycol (PEG), dextran, methylcellulose, polyvinyl alcohol (PVA), and polyvinyl pyrrolidone (PVP),caseinate, and alginate; salts, such as phosphates (e.g., tripotassiumphosphate and disodium phosphate), citrates (e.g., sodium citrate),sulfates (e.g., sodium sulfate), and carbonates. In some embodiments,one phase contains PEG and the other phase contains dextran.

The concentration of the solute can vary depending on the nature of thesolutes. Generally, the concentration is from about 3.5 wt % to thesolubility limited in water. In those embodiments where the solutes arePEG and dextran, the concentration of PEG is from about 3.5 wt % toabout 20 wt % and the concentration of dextran is from about 3.5 wt % tothe solubility limit of dextran.

The weight average molecular weight of PEG is from about 1,000 Daltonsto about 100,000 Daltons, preferably about 2,000 Daltons to about 20,000Daltons, preferably from about 2,000 Daltons to about 10,000 Daltons,more preferably from about 5,000 Daltons to about 10,000 Daltons. Insome embodiments, the molecular weight of PEG is about 8,000 Daltons.

The weight average molecular weight of dextran is from about 40,000Daltons to about 1,000,000 Daltons, preferably about 70,000 Daltons toabout 500,000 Daltons. In some embodiments, the molecular weight isabout 500,000 Daltons.

B. Therapeutic, Prophylactic, and Diagnostic Agents

One or more solutions can contain one or more therapeutic, prophylactic,and/or diagnostic agents. In some embodiments, the solution that formsthe dispersed phase contains one or more therapeutic, prophylactic,and/or diagnostic agents which are encapsulated within the droplets uponformation of the emulsion. The one or more therapeutic, prophylactic,and/or diagnostic agents can be small molecule therapeutic agents (e.g.,agents having molecular weight less than 2000 amu, 1500 amu, 1000, amu,750 amu, or 500 amu) and/or biomolecules, such as a proteins, nucleicacids (e.g., DNA, RNA, siRNA, etc.), enzymes, etc.

In some embodiments, the agent is a biomolecule, such as a protein,enzyme, nucleic acid, etc. Biomolecules can be denatured in the presenceof an organic solvent. Therefore, all aqueous emulsions provide avehicle for delivering such agents while preserving the biologicalactivity of the agent.

Agents to be delivered include therapeutic, nutritional, diagnostic, andprophylactic compounds. Proteins, peptides, carbohydrates,polysaccharides, nucleic acid molecules, and organic molecules, as wellas diagnostic agents, can be delivered. The preferred materials to beincorporated are drugs and imaging agents. Therapeutic agents includeantibiotics, antivirals (especially protease inhibitors alone or incombination with nucleosides for treatment of HIV or Hepatitis B or C),anti-parasites (helminths, protozoans), anti-cancer (referred to hereinas “chemotherapeutics”, including cytotoxic drugs such as doxorubicin,cyclosporine, mitomycin C, cisplatin and carboplatin, BCNU, 5FU,methotrexate, adriamycin, camptothecin, and taxol), antibodies andbioactive fragments thereof (including humanized, single chain, andchimeric antibodies), antigen and vaccine formulations, peptide drugs,anti-inflammatories, nutraceuticals such as vitamins, andoligonucleotide drugs (including DNA, RNAs, antisense, aptamers,ribozymes, external guide sequences for ribonuclease P, and triplexforming agents). Particularly preferred drugs to be delivered includeanti-angiogenic agents, antiproliferative and chemotherapeutic agentssuch as rampamycin.

Exemplary diagnostic materials include paramagnetic molecules,fluorescent compounds, magnetic molecules, and radionuclides.

Peptide, protein, and DNA based vaccines may be used to induce immunityto various diseases or conditions. For example, sexually transmitteddiseases and unwanted pregnancy are world-wide problems affecting thehealth and welfare of women. Effective vaccines to induce specificimmunity within the female genital tract could greatly reduce the riskof STDs, while vaccines that provoke anti-sperm antibodies wouldfunction as immunocontraceptives. Extensive studies have demonstratedthat vaccination at a distal site—orally, nasally, or rectally, forexample—can induce mucosal immunity within the female genital tract. Ofthese options, oral administration has gained the most interest becauseof its potential for patient compliance, easy administration andsuitability for widespread use. Oral vaccination with proteins ispossible, but is usually inefficient or requires very high doses. Oralvaccination with DNA, while potentially effective at lower doses, hasbeen ineffective in most cases because ‘naked DNA’ is susceptible toboth the stomach acidity and digestive enzymes in the gastrointestinaltract.

C. Cells

The capsules described herein are prepared from an aqueous dispersedphase and an aqueous continuous phase and therefore do not contain anyorganic solvents. Such capsules are desirable for encapsulating cells,the viability of which can be adversely affected by the organicsolvents. The cells can be added to the phase that becomes the coreand/or the phase that becomes the shell.

Exemplary cell types include, but are not limited to, kerotinocytes,fibroblasts, ligament cells, endothelial cells, lung cells, epithelialcells, smooth muscle cells, cardiac muscle cells, skeletal muscle cells,islet cells, nerve cells, hepatocytes, kidney cells, bladder cells,urothelial cells, stem or progenitor cells, neurobalstoma, chondrocytes,skin cells and bone-forming cells.

D. Emulsion Stabilization

Although the all-aqueous emulsions with controlled and tunablestructures have been generated with different approaches, all of theseemulsions are only transiently stable and tend to coalesce subsequently.Stabilization of these emulsions is thus useful in both scientificstudies and practical applications. In preferred embodiments, theemulsions are stabilized by adding a hydrogel-forming material to theshell-forming phase, forming the core-shell droplets, and solidifyingthe material to form capsules.

1. All-Aqueous Emulsion Templated Materials

Stabilized emulsion structures can be produced by selectivelysolidifying the dispersed phases, forming hydrogel beads or capsules. Toprevent the coalescence of droplets, photo-curable monomers such asPEGDA or dextran-HEMA can be added to the emulsion phase, and the fastphoto-polymerization helps to solidify the emulsion within seconds.However, photo-polymerization typically generates toxic free radicals,potentially harming the encapsulated species, especially the livingones. To achieve radical-free gelation, the emulsion phase can besolidified by diffusing biocompatible cross-linkers to the gelprecursors in the emulsion phase. For example, when a sodium alginatesolution is used as the emulsion phase, the emulsion can be quicklysolidified within a minute by introducing calcium ions. Leakage ofencapsulated species is negligible within the time scale of emulsiongelation. Nevertheless, many biocompatible gelation reactions, such asenzyme-induced gelation, last for hours to days, where leakage ofencapsulated species cannot be disregarded. In this manner, a compactmembrane must quickly form at the w/w interface, preventing the leakageof encapsulated species.

Suitable hydrogel forming materials include, but are not limited to,calcium alginate, polymer, such as modified PEGs (e.g., PEGdiacrylates), proteins, such as collagen or modified collagen, and blockcopolymers, such as PLURONICS®. In some embodiments, thehydrogel-forming materials are biocompatible. In other embodiments, thehydrogel-forming materials are biocompatible and biodegradable.

2. Water/Water Interface-Templated Materials

In other embodiments, the emulsion can be stabilized by forming amembrane or barrier on the surface of the dispersed phase droplets toprevent coalescence. Aggregation of particles or macromolecularsurfactants at the w/w interface is the primary mechanism of emulsiondestabilization. Submicron-sized latex microspheres and proteinparticles can be irreversibly trapped at the w/w interface. This featureindicates that the absorption energy is larger than the kinetic energyimposed by thermal activation. With a sufficiently large concentrationof protein particles and high w/w interfacial tension, protein particlessuccessfully stabilize the PEG/dextran emulsion for a few weeks.However, in the presence of a shear flow, these particles detach fromthe w/w interface and fail to stabilize the emulsion. Strengthening thebinding force among the protein particles may prevent the detachmentfrom the interfaces induced by the shear flow.

Self-assembly of macromolecules at the w/w interface provides anotherpossible solution to stabilize the all-aqueous emulsions. To form stableemulsions, macromolecular surfactants should aggregate at the w/winterface and form a compact membrane. Aggregation of the surfactants atthe w/w interface is strongly affected by their interactions with thedissolved solutes in aqueous phases. The presence of such interaction isconfirmed by the observation of budding of liposomes encapsulating twoimmiscible aqueous phases. In this example, two aqueous phasesselectively approach the different lipid domains after extraction ofwater from the liposomes. The interaction between the membrane and theincompatible solutes also keeps the membrane at the w/w interface. Thishypothesis is confirmed by using copolymers to form vesicles from thetemplates of w/w emulsions. In this study, the copolymers of thePEG-polycaprolactone (PCL) and the dextran-PCL are separately added intothe PEG-rich and the dextran-rich phase. Upon vortex mixing of the twophases, the two copolymers spontaneously aggregate at the w/w interface.More importantly, the PCL moieties facilitate the formation of a compactmembrane, probably due to the hydrophobic interactions.

E. Techniques for Manufacture

Techniques known in the art can be used to prepare the stabilizedemulsions described herein. In some embodiments, the emulsion isprepared using an electrospray technique.

Electrospray is a method of generating a very fine liquid aerosolthrough electrostatic charging. In electrospray, a liquid is passingthrough a nozzle. The plume of droplets is generated by electricallycharging the liquid to a very high voltage. The charged liquid in thenozzle becomes unstable as it is forced to hold more and more charge.Soon the liquid reaches a critical point, at which it can hold no moreelectrical charge and at the tip of the nozzle it blows apart into acloud of tiny, highly charged droplets. These tiny droplets aretypically less than 10 μm in diameter and fly about searching for apotential surface to land on that is opposite in charge to their own. Asthe droplets fly about, they rapidly shrink as solvent moleculesevaporate from their surface. Since it is difficult for charge toevaporate, the distance between electrical charges in the dropletdramatically decreases. If the droplet can't find a surface in which todissipate its charge in time, the electrical charge reaches a criticalstate and the droplet will violently blow apart again.

In the methods described herein, electrospray is used to contact a firstaqueous solution containing a first solute and a second aqueous solutioncontaining a second solute, wherein the solutes are incompatible. One ofthe solutions becomes the dispersed phase in the emulsion while theother becomes the continuous phase. The dispersed and continuous aqueousphase(s) are separated by a middle phase of air, preventing the mixingof charged solutes induced by high voltage. In some embodiments, adispersed phase (e.g., PEG-containing solution) is charged with a highDC voltage and is sprayed into an immiscible aqueous solution containingthe second solute (e.g., dextran) through air. The large surface tensionbetween the dispersed phase and air helps to break up the jet quicklyinto droplets. A dripping to jetting transition is observed upon anincrease in the applied voltage. In the dripping regime, the charged jetimmediately breaks up at the end of the spraying nozzle, yieldingmonodisperse droplets with a polydispersity of less than 5%, 4%, 3%, 2%,or 1%. In the jetting regime, polydisperse droplets are formed at theend of the Taylor cone. A reduction in the size of the spraying nozzlereduces the diameter of the jet, thus facilitating the fast formation ofdroplets in the electro-dripping regime.

The diameters of the dispersed droplets can be varied as a function ofthe applied voltage. For example, the diameter of PEG-droplets dispersedin a dextran continuous phase varied from about 800 microns to about 120microns by increasing the voltage from 4.2 kV to 5.0 kV. At thesevoltages, the droplets were monodisperse. Polydisperse droplets areobtained with further increases in the applied voltage.

A core-shell structured emulsion can be generated with the all-aqueouselectrospray approach. A round capillary with a tapered nozzle can becoaxially inserted into another tapered squared capillary, forming aco-flowing geometry. Two immiscible aqueous phases are separatelyinjected into the inner and outer glass capillaries, forming an innerphase-in-outer phase jet. The outer phase can be charged by ahigh-voltage power supply and the compound jet is forced to go through aring-shaped counter electrode under electrostatic forces. Upon breakupof the jet, core-shell structured droplets finally fall into thecontinuous phase. The relative sizes of the core and shell of theemulsion can be easily adjusted by changing the flow rates ratios of thetwo fluids. For example, varying the flow rate ratio of the shell (e.g.,PEG-rich phase) and core (e.g., dextran-rich phase) from 4:1 to 1:1 to1:5 resulted in an increase in the size of the core as shown by opticalimaging.

A core-shell structured emulsion can also be generated by takingadvantage of the phase separation of a single emulsion in an all-aqueouselectrospray approach. A single-phase jet containing two or moreincompatible solutes breaks up into single emulsion droplets viaall-aqueous electrospray. When the single emulsion droplets get into thecontinuous phase, an osmotic pressure between the two phases drives thecondensation of the emulsion phase. This leads to the phase separationof the two incompatible solutes in the droplet. Upon coalescence of thephase separated droplets, the single emulsion droplets are transformedinto core-shell structured emulsion droplets.

Core-shell structured capsules can be prepared by using the all-aqueousemulsion as templates. For example, to form hydrogel capsules from theemulsions described herein, a shell phase solution is preparedcontaining a hydrogel-forming material, such as sodium alginate and afirst solute, such as PEG. The core-shell structured droplets are thenproduced following the same procedure described above by contacting theshell phase solution with second solution (e.g., core solution)containing a second solute which is incompatible with the first soluteto form the all aqueous emulsion. In the case of sodium alginate, thedroplets are injected into solution containing calcium ions, such as acalcium chloride solution, forming calcium alginate capsules with theidentical sizes and geometrical features emulsion droplet template.

To form core-shell structured capsules, solidification of the shellphase can also by achieved by polymerization of the shell phasecontaining colloids or macromolecular monomers. For example, core-shellstructured protein capsules can be formed by dispersing protein-basedcolloids or monomers into the shell phase as the hydrogel formingmaterials. Suitable colloids include, but are not limited to,β-lactoglobulin (e.g., diameters ranging from 20 nm to 1000 nm), amyloidfibrils (e.g., length from 30 to 1000 nm), and collagen fiber (e.g. TypeI collagen from rat tail). Suitable protein-based monomers include, butare not limited to, lysozyme, albumin, insulin, and the like.

In some embodiments, the protein-based colloids and monomers can beinitially dispersed in a shell phase solution containing 4 wt %hydroxy-propyl methylcellulose solution. An immiscible aqueous phase,such as 10 wt % dextran, is used as the core liquid phase which forms anemulsion with the hydroxypropyl methylcellulose solution. The core-shellstructured droplets are produced following the same procedures describedabove. The yielded droplets are incubated, for example, at 65-90° C. formore than 24 hours or injected into a sodium chloride solution, formingprotein capsules with an aqueous core.

The diameter of the capsules can vary. In some embodiments, the capsuleshave an average diameter from about 500 nm to about 5 mm, preferablyfrom about 100 microns to about 5 mm. The diameter can be varied byvarying the applied electrical field, the diameter of the nozzle, and/orthe flow rate.

III. Applications

The micro- and/or nanocapsules described herein can be used forencapsulation applications known in the art, for example, for in vivodrug delivery, encapsulation of active agents for cosmetic applications,encapsulation of insecticides and/or herbicides, etc.

A. Drug Delivery

The emulsions described here can be used to deliver one or moretherapeutic, prophylactic, and/or diagnostic agent and/or cells to apatient in need thereof. As discussed above, the emulsions describedtherein do not contain an organic solvent and therefore are desirablefor encapsulating biomolecules (proteins, nucleic acids, etc.) and/orcells, which can be adversely affected by the presence organic solvents.Moreover, the presence of a membrane formed by the oppositely chargedmacromolecules improves the stability of the emulsions allowing them tobe prepared and stored for a period of time before use.

The emulsions can be formulated for a variety of routes ofadministration. In some embodiments, the emulsion is administeredenterally (e.g., oral) or parenterally.

“Parenteral administration”, as used herein, means administration by anymethod other than through the digestive tract or non-invasive topical orregional routes. For example, parenteral administration may includeadministration to a patient intravenously, intradermally,intraarterially, intraperitoneally, intralesionally, intracranially,intraarticularly, intraprostatically, intrapleurally, intratracheally,intravitreally, intratumorally, intramuscularly, subcutaneously,subconjunctivally, intravesicularly, intrapericardially,intraumbilically, by injection, and by infusion.

The emulsions can be administered neat, i.e., without additionalcarriers/excipients. Alternatively, the emulsions can be combined withone or more carriers and/or excipients to prepare a pharmaceuticalcomposition.

The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, one or more polyols (e.g., glycerol, propyleneglycol, and liquid polyethylene glycol), oils, such as vegetable oils(e.g., peanut oil, corn oil, sesame oil, etc.), and combinationsthereof. The proper fluidity can be maintained, for example, by the useof a coating, such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and/or by the use ofsurfactants. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride.

Suitable surfactants may be anionic, cationic, amphoteric or nonionicsurface active agents. Suitable anionic surfactants include, but are notlimited to, those containing carboxylate, sulfonate and sulfate ions.Examples of anionic surfactants include sodium, potassium, ammonium oflong chain alkyl sulfonates and alkyl aryl sulfonates such as sodiumdodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodiumdodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodiumbis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodiumlauryl sulfate. Cationic surfactants include, but are not limited to,quaternary ammonium compounds such as benzalkonium chloride,benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzylammonium chloride, polyoxyethylene, and coconut amine Examples ofnonionic surfactants include ethylene glycol monostearate, propyleneglycol myristate, glyceryl monostearate, glyceryl stearate,polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG-150laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates,polyoxyethylene octylphenylether, PEG-1000 cetyl ether, polyoxyethylenetridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401,stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallowamide. Examples of amphoteric surfactants include sodiumN-dodecyl-β-alanine, sodium N-lauryl-β-iminodipropionate,myristoamphoacetate, lauryl betaine, and lauryl sulfobetaine.

The formulation can contain a preservative to prevent the growth ofmicroorganisms. Suitable preservatives include, but are not limited to,parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. Theformulation may also contain an antioxidant to prevent degradation ofthe active agent(s).

The formulation is typically buffered to a pH of 3-8 for parenteraladministration upon reconstitution. Suitable buffers include, but arenot limited to, phosphate buffers, acetate buffers, and citrate buffers.

Water soluble polymers are often used in formulations for parenteraladministration. Suitable water-soluble polymers include, but are notlimited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, andpolyethylene glycol.

Enteral formulations are prepared using pharmaceutically acceptablecarriers. As generally used herein “carrier” includes, but is notlimited to, diluents, preservatives, binders, lubricants,disintegrators, swelling agents, fillers, stabilizers, and combinationsthereof. Polymers used in the dosage form include hydrophobic orhydrophilic polymers and pH dependent or independent polymers. Preferredhydrophobic and hydrophilic polymers include, but are not limited to,hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethylcellulose, carboxy methylcellulose, polyethylene glycol, ethylcellulose,microcrystalline cellulose, polyvinyl pyrrolidone, polyvinyl alcohol,polyvinyl acetate, and ion exchange resins.

Carrier also includes all components of the coating composition whichmay include plasticizers, pigments, colorants, stabilizing agents, andglidants. Formulations can be prepared using one or morepharmaceutically acceptable excipients, including diluents,preservatives, binders, lubricants, disintegrators, swelling agents,fillers, stabilizers, and combinations thereof.

Delayed release dosage formulations can be prepared as described instandard references such as “Pharmaceutical dosage form tablets”, eds.Liberman et al. (New York, Marcel Dekker, Inc., 1989), “Remington—Thescience and practice of pharmacy”, 20th ed., Lippincott Williams &Wilkins, Baltimore, Md., 2000, and “Pharmaceutical dosage forms and drugdelivery systems”, 6th Edition, Ansel et al., (Media, Pa.: Williams andWilkins, 1995). These references provide information on excipients,materials, equipment and process for preparing tablets and capsules anddelayed release dosage forms of tablets, capsules, and granules. Thesereferences provide information on carriers, materials, equipment andprocess for preparing tablets and capsules and delayed release dosageforms of tablets, capsules, and granules.

Stabilizers are used to inhibit or retard drug decomposition reactionswhich include, by way of example, oxidative reactions. Suitablestabilizers include, but are not limited to, antioxidants, butylatedhydroxytoluene (BHT); ascorbic acid, its salts and esters; Vitamin E,tocopherol and its salts; sulfites such as sodium metabisulphite;cysteine and its derivatives; citric acid; propyl gallate, and butylatedhydroxyanisole (BHA).

EXAMPLES Example 1: Preparation of all Aqueous Emulsions UsingElectrostatic Effects

A dispersed phase of 8 wt % PEG (Mw=8,000) solution charged with a highDC voltage is sprayed into its immiscible aqueous phase of 15 wt %dextran (Mw=500,000) solution through air. The large surface tensionbetween the dispersed phase and air helps to break up the jet quicklyinto droplets (FIG. 1, panel a). A dripping to jetting transition isobserved upon an increase in the applied voltage. In the drippingregime, the charged jet immediately breaks up at the end of the sprayingnozzle, yielding monodisperse droplets with a polydispersity of lessthan 5% (FIG. 1, panel b). By increasing the applied electrical fieldfrom 2.1 kV/cm to 2.5 kV/cm, the diameter of the produced droplets issignificantly reduced from 810 μm to 120 μm (FIG. 2). In this case, thedistance between the positively charged nozzle and the negativelycharged electrode ring is 2 cm, and the diameter of the nozzle is 40micrometers. Further increase in the strength of the electric fieldleads to polydisperse droplets with smaller sizes. A reduction in thesize of the spraying nozzle can produce monodisperse droplets withsmaller sizes. For example, when the size of nozzle is decreased to 20μm, uniform droplets with diameters of less than 50 μm are produced.

Example 2: Preparation of Core-Shell all Aqueous Emulsions UsingElectrostatic Effects

Two immiscible aqueous phases of 10% dextran (Mw=500,000) and 8 wt % PEG(Mw=8,000) solutions were separately injected into inner and outer glasscapillaries, forming a dextran-in-PEG jet (FIG. 1, panel a). ThePEG-rich phase was charged by a high-voltage power supply and thecompound jet was forced to go through a ring-shaped counter electrodeunder electrostatic forces. Upon breakup of the jet, core-shellstructured droplets fell into the continuous phase of a dextran solutionor on the surface of a solid substrate (FIG. 1, panel c). The diameterof the core was varied by varying the flow rate ratio of the PEG-rich(shell) and dextran-rich (core) phase. As the ratio was varied from 4:1to 1:1 to 1:5, the diameter of the core increased.

Core-shell structured capsules can be prepared by using the all-aqueousemulsion as templates. For example, to form calcium alginate hydrogelcapsules from the above emulsions, 1 wt %-4 wt % sodium alginate isdissolved into 8% PEG as the shell liquid phase. The core-shellstructured droplets are then produced following the same proceduresdescribed above. The droplets are injected into 2 wt-8 wt % calciumchloride solutions, forming calcium alginate capsules with the identicalsizes and geometrical features to the template of emulsion droplets.

Protein-based colloids and monomers can be initially dispersed in ashell phase solution containing 4 wt % hydroxy-propyl methylcellulosesolution. An immiscible aqueous phase, such as 10 wt % dextran, is usedas the core liquid phase which forms an emulsion with the hydroxypropylmethylcellulose solution. The core-shell structured droplets areproduced following the same procedures described above. The yieldeddroplets are incubated, for example, at 65-90° C. for more than 24 hoursor injected into a sodium chloride solution, forming protein capsuleswith an aqueous core.

Example 3: Preparation of Core-Shell all Aqueous Emulsions UsingElectrostatic Effects

A single-phase solution dissolved with 5% dextran (Mw=500,000) and 1%PEG (Mw=20,000) are injected into a glass capillary. This solution wascharged by a high-voltage power supply and the compound jet was forcedto go through a ring-shaped counter electrode under electrostaticforces. Upon breakup of the jet, single emulsion droplets fell into thecontinuous phase of 8% PEG (Mw=8,000). Due to osmotic pressure betweenthe two phases, water is gradually extracted from the droplet phase. Thecondensation of the droplet results in a phase separation inside thedroplets, yielding a dextran-rich shell and PEG-rich liquid core (FIG.3). The diameter of the core was varied by varying the concentration ofPEG and dextran in the dispersed phase. As the concentration ratio ofdextran and PEG was varied from 4:1 to 10:1, the volume ratio of theshell and core phases decreased accordingly.

Core-shell structured capsules can be prepared by using the all-aqueousemulsion as templates. For example, 1 mg/ml-5 mg/ml collagen, type I isdissolved into the single-phase solution of 5% dextran and 1% PEG. Afterformation of core-shell structured emulsion, collagen accumulates intothe dextran-rich shell. Subsequent solidification of the collagen,either by heating at 37-60° C. for 8-24 hours or chemical cross-linkingby 2% glutaraldehyde for 2 hours can lead to the formation of core-shellstructured capsules.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Publications cited herein andthe materials for which they are cited are specifically incorporated byreference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

We claim:
 1. A biocompatible capsule comprising (a) a liquid core, and(b) a solid or semi-solid shell comprising sodium alginate, or calciumalginate, or collagen, wherein the core contains a first solutecomprising polyethylene glycol (PEG) and the solid or semi-solid shellcomprises a hydrogel comprising dextran; or the core contains a firstsolute comprising dextran and the solid or semi-solid shell comprises ahydrogel comprising PEG; and wherein the core, the shell, or bothcomprises one or more therapeutic, prophylactic, diagnostic agents,and/or cells.
 2. The biocompatible capsule of claim 1, wherein the firstsolute further comprises methyl cellulose, polyvinyl alcohol, polyvinylpyrrolidone; tripotassium phosphate, sodium citrate, sodium sulfate, ordisodium phosphate.
 3. The biocompatible capsule of claim 1, wherein themolecular weight of dextran is about 500,000 Daltons.
 4. Thebiocompatible capsule of claim 1, wherein the molecular weight of PEG isabout 8,000-20,000 Daltons.
 5. The biocompatible capsule of claim 1,wherein the one or more cells comprise kerotinocytes, fibroblasts,ligament cells, endothelial cells, lung cells, epithelial cells, smoothmuscle cells, cardiac muscle cells, skeletal muscle cells, islet cells,nerve cells, hepatocytes, kidney cells, bladder cells, urothelial cells,stem or progenitor cells, neuroblastoma, chondrocytes, skin cells orbone-forming cells.
 6. The biocompatible capsule of claim 1, wherein thecore is an aqueous liquid core, the shell is a hydrogel, and the capsuleis free of an organic solvent.
 7. The biocompatible capsule of claim 1,wherein the capsule is free of a surfactant.
 8. The biocompatiblecapsule of claim 1, wherein the first solute comprises dextran andwherein the concentration of dextran in the first solute is about 5-15%.9. The biocompatible capsule of claim 1, wherein the first solutecomprises PEG, and wherein the concentration of PEG in the first soluteis 8%.
 10. The biocompatible capsule of claim 1 prepared by a methodcomprising (a) forming a coaxial jet comprising an electrically chargedaqueous core phase containing the first solute and an optionally chargedaqueous shell phase containing a shell forming material and a secondsolute which is incompatible with the first solute; (b) breaking up thejet to form core-shell structured droplets; and (c) solidifying thedroplets to form the capsules.
 11. The biocompatible capsule of claim10, wherein the diameter of the capsule is from about 500 nm to about 5mm.
 12. A method for administering one or more therapeutic,prophylactic, and/or diagnostic agent to a patient in need thereof, themethod comprising administering the biocompatible capsules of claim 1.13. The method of claim 12, wherein the agent is a biomolecule, a smallmolecule, or combinations thereof.
 14. The method of claim 12, whereinthe capsules are administered orally.
 15. The method of claim 12,wherein the capsules are administered parenterally.
 16. A method foradministering cells to a patient in need thereof, the method comprisingadministering the biocompatible capsules of claim
 1. 17. The method ofclaim 16, wherein the capsules are administered parenterally.